Journal of Controlled Release 107 (2005) 229 – 243

www.elsevier.com/locate/jconrel

Saturated phospholipids promote crystallization but slow down

polymorphic transitions in triglyceride nanoparticles
Heike Bunjes a,*, Michel H.J. Koch b
a
Friedrich Schiller University Jena, Institute of Pharmacy, Department of Pharmaceutical Technology, Lessingstrasse 8,
D-07743 Jena, Germany
b
European Molecular Biology Laboratory, Hamburg Outstation, EMBL c/o DESY, Notkestrasse 85, D-22603 Hamburg, Germany
Received 16 March 2005; accepted 13 June 2005
Available online 14 July 2005

Abstract

Some matrix materials proposed for the preparation of solid lipid nanoparticles (e.g. trilaurin) are difficult to crystallize after
processing by melt-homogenization. In an attempt to overcome this difficulty, the effect of saturated long-chain phospholipids
on the crystallization of nanoparticles based on trilaurin, trimyristin, tripalmitin and tristearin was studied. The phospholipids
were used as emulsifiers in combination with sodium glycocholate. Saturated phospholipids increased the crystallization
temperature of the triglyceride by several degrees compared to soybean phospholipids. The crystallization pattern was more
complex in such systems due to solidification of the phospholipid chains prior to triglyceride crystallization. For most
triglycerides, egg lecithin also induced crystallization at higher temperatures than natural soybean lecithin. With trilaurin
dispersions, the effect of phospholipids can be utilized to induce crystallization at temperatures relevant for larger scale
preparation. The polymorphic transitions of the triglycerides were slower in the presence of egg and saturated lecithin leading to
a higher stability of the metastable a-form. These effects were particularly pronounced in tristearin systems where a
predominant fraction of a-phase particles could be observed even after long-term cold storage in dispersions containing
hydrogenated soybean lecithin or DPPC. The possibility to prepare triglyceride nanoparticles stable in specific modifications
offers new opportunities to study effects of polymorphic form on colloidal stability, drug loading and release properties of such
dispersions.
D 2005 Elsevier B.V. All rights reserved.

Keywords: Colloidal drug carriers; Solid lipid nanoparticles; Triglycerides; Crystallization in emulsions; Polymorphic transitions

1. Introduction

Solid lipid nanoparticles, e.g., based on triglycer-

* Corresponding author. Tel.: +49 3641 949903; fax: +49 3641
949902.
E-mail address: Heike.Bunjes@uni-jena.de (H. Bunjes).
0168-3659/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.jconrel.2005.06.004
ides, waxes or fatty acids as matrix lipids, are being
intensively investigated as potential drug carrier sys-
tems, in particular for lipophilic substances [1]. Their
230 H. Bunjes, M.H.J. Koch / Journal of Controlled Release 107 (2005) 229–243
small particle size makes them interesting as delivery
systems for different modes of administration, includ-
ing the intravenous route. Colloidal dispersions of
solid lipid particles can be obtained by different meth-
ods, for example by high-pressure homogenization of
a hot premix with subsequent crystallization of the
dispersed lipids or by precipitation from hot micro-
emulsions [1]. Some of the matrix materials proposed
for use in such dispersions (e.g., trilaurin) are, how-
ever, difficult to crystallize after processing into nano-
particles via a melt-dispersion process due to a
pronounced depression of the crystallization temper-
ature (T C) in the colloidal particles [2,3]. The resulting
dispersions can remain as emulsions of supercooled,
liquid droplets for months or even years if no special
measures are taken to crystallize the nanoparticles
[4,5]. It is important to find solutions to this problem
as the proposed advantages of solid lipid nanoparti-
cles, e.g., stronger immobilization of incorporated
drugs, enhanced surface modification potential or sta-
bility against coalescence, rely on the presence of a
solid particle core. This is especially relevant for
trilaurin dispersions which are interesting candidates
for the processing of thermolabile substances and the
formulation of temperature-sensitive drug delivery
systems due to the low melting point of this material
around 46 8C.
One way to increase the T C of nanoparticles
prepared from shorter-chain triglycerides is to add
longer-chain triglycerides like tristearin to the matrix
composition. Unfortunately, this also alters the melt-
ing properties of the nanoparticle matrix and may
lead to a more complex crystallization behavior [2].
Stabilization with classical surfactants (e.g., Tween,
Brij) containing longer, saturated alkyl chains also
helps to overcome the crystallization problem [6,7].
The use of such surfactants as the only stabilizers
for triglyceride nanodispersions, however, often neg-
atively affects the colloidal quality of the disper-
sions [6–8]. Phospholipids have frequently been
used with good results in stabilizer compositions
for solid lipid nanoparticle dispersions. The question
arises, thus, as to whether the use of long-chain
saturated phospholipids can increase the crystalliza-
tion tendency of colloidal droplets of different sat-
urated triglycerides while avoiding the problems
with colloidal stability. Therefore, the effect of dif-
ferent phospholipids on the crystallization behavior
of triglyceride nanoparticles was compared to that
of the mainly unsaturated soybean phospholipids
commonly employed for lipid nanoparticle stabiliza-
tion. Since the stabilization of triglyceride nanopar-
ticles with phospholipids only usually leads to the
formation of highly viscous or gel-like systems
upon triglyceride crystallization [9] the dispersions
were co-stabilized with the bile salt sodium glyco-
cholate as a physiologically acceptable surfactant in
combination with phospholipids. In addition to their
potential influence on the T C surface active agents
may also influence the crystalline structures formed
in solid lipid nanoparticles as previously observed
for melt-homogenized triglyceride nanoparticles [6,
8] and for stearic acid nanoparticles prepared by the
microemulsion method [10–12]. A potential influ-
ence of the different phospholipids on the polymor-
phism of the dispersed triglycerides was thus also
taken into consideration.
2. Materials and methods
2.1. Materials
The triglycerides trilaurin (Dynasan 112, D112),
trimyristin (Dynasan 114, D114) tripalmitin (Dynasan
116, D116) and tristearin (Dynasan 118, D118) were
kindly provided by Condea Chemie, D-Witten.
According to manufacturerTs specification the purity
of the fatty acid fraction is about 95% (D114, D116) or
99% (D112, D118), respectively, for these triglyceride
qualities. The hydroxyl value indicating the content of
partial glycerides is below 5 in all cases. The phospho-
lipids Lipoid S100 (S100), Lipoid S75 (S75) (soybean
lecithins with different grades of purification), Lipoid
S100-3 (S100-3), Lipoid S75-3 (S75-3) (corresponding
hydrogenated lecithins), Lipoid E100 (E100), Lipoid
E80 (E80) (egg yolk lecithins with different grades of
purification) were a kind gift of Lipoid, D-Ludwigsha-
fen. Some relevant specifications for these mixed chain
phospholipids are listed in Table 1. Dimyristoylpho-
sphatidylcholine (DMPC) and dipalmitoylphosphati-
dylcholine (DPPC) were donated by Astra Arcus (S-
Södertälje). The lipids as well as sodium glycocholate
(SGC, Sigma-Aldrich Chemie, D-Taufkirchen), glyc-
erol (Solvay Chemicals, D-Hannover) and thiomersal
(Synopharm, D-Barsbüttel) were used as received. The
 H. Bunjes, M.H.J. Koch / Journal of Controlled Release 107 (2005) 229–243 231

Table 1
Phosphatidylcholine content and typical fatty acid composition of phospholipids from natural sources (manufacturer’s specifications)
S75 S75-3 S100 S100-3 E80 E100
Phosphatidylcholine ( in % ):
68–73 (incl. LPC) 67–71 (incl. LPC) z94 z94 80–85 z94

Typical fatty acid composition (in % of total fatty acids):

C16: 0 17–20 12–16 12–17 12–16 28–34 30–33
C16: 1 V3
C18: 0 2–5 80–85 2–5 85–88 12–15 11–15
C18: 1 8–12 V5 11–15 V2 26–30 27–32
C18: 2 58–65 V2 59–70 V1 13–18 14–18
C18: 3 4–6 3–7 V1
C20: 0 3–4
C20: 1 + n and higher 6–10 2–5
LPC: Lysophosphatidylcholine.

water for the dispersions was purified by reverse os-
mosis (Alpha Q, Direct Q 5, Millipore, D-Schwalbach).
3. Methods
3.1. Preparation of the dispersions
The dispersion composition was chosen based on
previous good experience with solid triglyceride/soy-
bean phospholipid/sodium glycocholate mixtures re-
garding the formation of nanoparticle dispersions with
adequate particle size and long term stability
[5,8,9,38]. The dispersions were prepared from 10%
triglyceride, 2.4% phospholipid and 0.6% sodium
glycocholate (w/w concentrations) in an aqueous
phase containing 2.25% glycerol and 0.01% thiomer-
sal (w/v concentrations). Sample names used in the
text refer to the matrix lipid and stabilizer composi-
tion: e.g., D118-S100/SGC is a tristearin dispersion
stabilized with a combination of Lipoid S100 and
sodium glycocholate. Phospholipid and glycocholate
were dispersed/dissolved in the aqueous phase by
magnetic stirring overnight; dispersions containing
phospholipids with a phase transition above room
temperature were transiently heated above the phase
transition temperature to facilitate dispersion. The
stabilizer dispersion was heated to the respective prep-
aration temperature (~65 8C for D112, ~75 8C for
D114, ~85 8C for D116, ~90 8C for D118) which
was always above the phase transition temperature of
the incorporated phospholipid. The hot liquid was
added to the molten triglyceride which had been
heated to the same temperature and the mixture was
predispersed by probe sonication (Bandelin Desinte-
grator HD200, D-Berlin). The hot predispersion was
passed through a heated high pressure homogenizer
(Micron Lab 40, APV Gaulin, D-Lübeck) for 5 cycles
at 800 bars and filled into glass vials. The D112
dispersions were allowed to cool down passively to
room temperature after homogenization. The other
dispersions were subjected to controlled cooling in a
thermostat at 0.5 8C/min from 60 to 5 8C (D114), from
60 to 18 8C (D116) or from 65 to 23 8C (D118).
Fractions of the samples were stored at 238C and at
refrigerator temperature (~2–8 8C). For comparison,
triglyceride-free dispersions were prepared in a similar
manner (2.67% phospholipid, 0.67% sodium glyco-
cholate, homogenization at ~2–8 8C); these homoge-
nized dispersions were passively cooled to room
temperature. To check the influence of the type of
phospholipid on trilaurin crystallization under isother-
mal conditions small amounts of the D112 dispersions
were stored at 0 8C for 24 h in a thermostat. At this
temperature, the aqueous phase does not crystallize
due to the presence of glycerol.
3.2. Particle size analysis
Particle sizes were measured by photon correlation
spectroscopy at 25 8C and at a scattering angle of
1738 (Zetasizer Nano-ZS, Malvern Instruments, D-
Herrenberg). Prior to measurement the samples were
diluted with demineralized, dust-free water to an al-
most clear optical appearance. The mean particle size
was estimated by analyzing the data with the cumu-
232 H. Bunjes, M.H.J. Koch / Journal of Controlled Release 107 (2005) 229–243
lants method assuming spherical particles. The results
are given as the z-average diameter (an intensity
weighted mean diameter) and the polydispersity
index (PI) as a measure for the relative width of the
particle size distribution. Values given are means of 3
successive 5 min measurement runs after 5 min equil-
ibration. Additional measurements were performed on
trimyristin, tripalmitin and tristearin dispersions cold
stored for several months (D114: ~ 4 months, D116:
~12 months, D118: ~10 months) with a Coulter LS
230 Particle Sizer (Beckman Coulter, D-Karlsruhe)
combining laser diffraction and polarization intensity
differential scattering (PIDS) methods. Values given
are the average of 8 successive 90 s runs. Size dis-
tributions by volume were calculated applying an
optical model created by the instrumental software
assuming a refractive index of 1.54 for the solid
lipid particles.
3.3. Differential scanning calorimetry
Thermograms were recorded with a Pyris 1 DSC
(Perkin Elmer). About 10–15 mg of dispersion were
accurately weighed into standard aluminium pans
which were tightly sealed. For the investigation of
bulk material, about 1 – 2 mg of sample were used.
An empty pan was used as reference. Samples were
heated at 5 or 10 8C/min, cooling was performed at
5 8C/min (note that the T Cs obtained at this compar-
atively high cooling rate are lower than those under
isothermal conditions). T Cs are given as the extrapo-
lated peak onset, melting temperatures as the peak
maximum temperature. The values obtained for the
melting enthalpy are only approximate since the not
entirely quantitative preparation technique may lead to
slight variations in sample composition. To calculate
the amount of recrystallized triglyceride after storage
of differently stabilized trilaurin dispersions at 0 8C
the melting enthalpy of the original sample was com-
pared with that of the same sample after cooling to
10 8C for 5 min to fully crystallize the triglyceride.
The values for the melting enthalpy of the h-form in
Fig. 5 represent the area under the endothermic peak
at high temperatures (cf. Fig. 4). When an exothermic
event due to recrystallization of a small amount of
material into the stable modification directly preceded
this melting endotherm, the transition enthalpy of this
exotherm was subtracted from that of the h-melting
endotherm in order to obtain the melting enthalpy of
the h-form originally present in the sample. The
resulting values also represent approximations as
this method has some uncertainties, due to e.g., ab-
sence of baseline separation from the a-melting en-
dotherm, a very structured thermal event without
distinct assignment of the single transitions, potential
overlap with phospholipid phase transitions and in-
clusion of h’-form in the melting peak at higher
temperature as well as a preparative procedure that
potentially leads to slight differences in triglyceride
content.
3.4. X-ray diffraction
Simultaneous small and wide angle synchrotron
radiation X-ray diffraction patterns were recorded
with two linear delay line detectors on the X33 camera
of the EMBL on the storage ring DORIS of the
Deutsches Elektronen Synchrotron (DESY) [13,14].
Data reduction and correction for detector response
were done with the program SAPOKO [15]. The
sample cells were thermostated with a waterbath
(Huber Ministat) controlled via an IF232 programma-
ble interface. For recrystallization studies, the nano-
particles were kept at 70 8C for about 10 min and then
rapidly cooled to 21 8C (D114-S100/SGC) or 47 8C
(D114-DPPC/SGC). The samples were further cooled
at about 0.3 8C/min and the scattering pattern was
monitored continuously in 1 min time frames. The
graphs shown in Fig. 6 represent the average of three
1-min exposures around the temperature given in the
diagrams.
4. Results
4.1. Particle size and macroscopic appearance of the
dispersions
All triglyceride dispersions were macroscopically
homogenous milky systems with PCS z-average par-
ticle sizes between about 130 and 180 nm and poly-
dispersity indices between 0.11 and 0.21 after cold
storage (Fig. 1). No meaningful PCS measurement
could be performed on the cold stored D116-S100-3/
SGC dispersion due to its tendency to gel upon shear
stress. No such tendency was, observed, however, in
 H. Bunjes, M.H.J. Koch / Journal of Controlled Release 107 (2005) 229–243 233

Trilaurin Trimyristin
250

Polydispersity index
0.2
200
Particle size [nm]
0.1
150 0.0

100

50

0
S100 E100 S100-3 DMPC DPPC S100 E100 S100-3 DMPC DPPC

Tripalmitin Tristearin
250

Polydispersity index
0.2
200
Particle size [nm]

0.1
150 0.0

100

50

0
S100 E100 S100-3 DMPC S100 E100 S100-3 DMPC DPPC
S75 E80 S75-3 DPPC

Fig. 1. PCS z-average mean particle sizes (bars) and polydispersity indices (diamonds) of the dispersions (cold stored, except for the D116-
S100-3/SGC dispersion). D112-particle sizes were obtained on dispersions stored at 0 8C to ensure particle crystallization (D112-S100/SGC still
does not crystallize under these conditions). All graphs are on the same scale.

the corresponding sample stored at room temperature.
Evaluation of trimyristin, tripalmitin and tristearin dis-
persions with laser diffraction after several months of
cold storage usually yielded median particle sizes
between about 120 and 180 nm with 99% of particle
sizes below a value (d99) around 300 nm. The D116
dispersions containing S100-3 and DPPC which had
turned into highly viscous systems during storage dis-
played median values around 47 or 19 Am, respective-
ly. Measurements on the corresponding systems stored
at room temperature revealed, however, median and
d99 values in the usual range also for these two dis-
persions. For the S100-containing D116 and D118
dispersions, the d99 values were in the Am-range but
more than 98% of the material still had sizes below 1
Am. These high values were, however, not confirmed
for the corresponding dispersions stored at 23 8C
(d99 b 300 nm). The E100-containing D116 dispersion
had a d99 value between 740 and 750 nm in the cold
stored sample as well as in a control measurement on
the sample stored at room temperature. At the time of
the laser diffraction measurements, dispersions stabi-
lized with natural phospholipids had a slightly (S100)
to distinctly (E100) yellowish color whereas systems
with saturated phospholipids were milky white.
4.2. Crystallization temperature (T C)
Tristearin, tripalmitin and trimyristin formed solid
nanoparticles under the chosen preparative conditions
as reflected in characteristic DSC melting transitions
around ~68–69 8C (tristearin), ~58 8C (tripalmitin)
and ~53–54 8C (trimyristin). Trilaurin particles,which
would melt around 42–43 8C in the crystalline state,
remained, however, liquid during storage at room
temperature: even after more than one year no or
less than 0.5% recrystallized triglyceride was detected
by DSC. All dispersed triglycerides crystallized at
temperatures much below their melting point reflect-
ing the high supercooling tendency of these materials
in the colloidal state (Fig. 2). In agreement with
previous investigations the T C decreased with the
chain length of the triglyceride [2] but it also
depended on the type of phospholipid in the disper-
234 H. Bunjes, M.H.J. Koch / Journal of Controlled Release 107 (2005) 229–243

57-31ºC
15ºC 48ºC 29ºC 35ºC 60ºC 50ºC
10 30

Temperature [ºC]
Temperature [ºC]

5 25

0 20

-5 15

Trilaurin (C12) Trimyristin (C14)

-10 10
S100 E100 S100-3 DPPC DMPC S100 DMPC E100 S100-3 DPPC

40ºC 49ºC 62ºC 61ºC 53ºC 52ºC 63ºC

40 50

Temperature [ºC]
Temperature [ºC]

35 45

41ºC
30 40

25 35

Tripalmitin (C16) Tristearin (C18)

20 30
S100 E100 E80 S100-3 S100 E100 DMPC DPPC S100-3
S75 DMPC DPPC S75-3

Fig. 2. Temperatures of the main crystallization event (diamonds) and temperature range of the precrystallization exotherm (dashed arrows and
attached temperature values). The thermal event at high temperature in the S100-containing tristearin dispersion is probably caused by trace
amounts of coarser triglyceride material. For samples containing S100, S75, E100 or E80 the small solid arrows indicate the approximate range
of the first deviation of the DSC trace from the baseline.

sion. Stabilization with soybean lecithin (S100, S75) stearin nanoparticles, E100, which was as effective
led to the lowest T C for all triglycerides. The use of as S100-3 in trilaurin, did not increase the T C com-
egg yolk lecithin (E100, E80) or fully saturated phos- pared to S100. The difference between the lowest and
pholipids (S100-3, S75-3, DMPC, DPPC) tended to the highest T C within a series of a given triglyceride
increase the T C. Compared to soybean lecithin all increased from trilaurin (DT = ~6 8C) over trimyristin
other investigated phospholipids significantly, but (DT = ~7 8C) to tripalmitin and tristearin particles
nearly independently of their exact nature, increased (DT = ~8 8C). Except in dispersions stabilized with
the T C of trilaurin. Storage of trilaurin dispersions for egg lecithin crystallization at higher temperatures was
24 h at 08C led to virtually complete crystallization always preceded by a complex exothermal event as
(~96–101% in DSC) of the triglyceride in the pres- indicated in Fig. 2 (dashed lines) and exemplified in
ence of E100, S100-3, DMPC and DPPC. In contrast, Fig. 3. These bprecrystallizationQ events were, in most
the dispersion with S100 contained only about 1% cases, not clearly separated from the main crystalliza-
recrystallized triglyceride after this thermal treatment. tion event and had different sizes and shapes. The
For trimyristin nanoparticles DMPC and E100 shifted onset temperature of precrystallization tended to in-
the T C to a higher value compared to S100 but they crease for a given phospholipid with the chain length
were less effective in increasing the temperature than of the triglyceride in the dispersion. This effect was
S100-3 and DPPC. For dispersions of tripalmitin and particularly obvious in DMPC-stabilized dispersions.
tristearin the saturated soybean phospholipids (S100-3, Exothermal events were also observed upon cool-
S75-3) led to the highest T Cs, whereas the other ing the triglyceride-free dispersions containing S100-3
phospholipids induced intermediate values. For tri- (~57–24 8C, maximum at 49.8 8C), DPPC (~49–27 8C,
 H. Bunjes, M.H.J. Koch / Journal of Controlled Release 107 (2005) 229–243 235

"Precrystallization"
maxima at 42.3 and 34.9 8C) and DMPC (~31–5 8C,
Cooling
maxima at 21.5 and 10.9 8C) in the temperature range
between 85 and 10 8C but not in the samples with
Heat Flow

S100 and E100. The transition enthalpy was, howev-

er, much smaller for these events (approximately be-
2 mW
tween 0.8 and 1.6 J/g) than that for the overall
exotherms of the triglyceride dispersions (between
about 8.5 and 17 J/g). It is difficult to compare
Triglyceride crystallization
the enthalpy of transition of the precrystallization in
0 10 20 30 40 50 60 70 triglyceride dispersions and of the thermal event in the
Temperature [ºC] triglyceride-free systems. The precrystallization event
Fig. 3. DSC cooling curve of the D116-DPPC/SGC dispersion usually overlaps with the main crystallization of the
exemplifying the structured thermal event upon cooling observed triglyceride and cannot accurately be evaluated. A
in dispersions stabilized with saturated phospholipids. coarse estimate of the transition enthalpy during the

Trilaurin (C12) Trimyristin (C14)

5 mW
Heat flow 5 mW

DPPC S100-3
Heat flow

DPPC
S100-3
E100
DMPC
DMPC
E100
S100 S100

0 10 20 30 40 50 60 70 10 20 30 40 50 60 70 80
Temperature [ºC] Temperature [ºC]

Tripalmitin (C16) Tristearin (C18)

5 mW

5 mW

DPPC
S75-3
Heat flow

S100-3
Heat flow

S100-3
DPPC
E80
E100 E100
DMPC
DMPC
S75
S100 S100

30 40 50 60 70 80 90 30 40 50 60 70 80 90
Temperature [ºC] Temperature [ºC]

Fig. 4. DSC heating curves (10 8C/min; 5 8C/min for trilaurin) of the dispersions after controlled cooling of the freshly prepared systems and 1 h
storage at room temperature (tripalmitin, tristearin) or directly after crystallization in the DSC (trilaurin, trimyristin). The dashed vertical lines
indicate the approximate temperature expected for the melting point of the a-modification [40].
236 H. Bunjes, M.H.J. Koch / Journal of Controlled Release 107 (2005) 229–243

20
pretransition and comparison with that of the pure
18
phospholipid dispersions revealed smaller as well as

Transition enthalpy [J/g]

16
larger transition enthalpies for the precrystallization
14
event. As a trend, the precrystallization event seems to 12 S100
become larger than in the corresponding phospholip- 10
S100 cs
DMPC
id/bile salt dispersions when the acyl chain length of 8 E100
the triglyceride approaches or exceeds that of the 6 E100 cs
phospholipid. S100-3
4
S100-3 cs
2 DPPC
Tripalmitin (C16)
4.3. Polymorphism 0
0 1 2 3 4 5 6 7
Days after preparation
In DSC, dispersions of freshly crystallized nano-
20
particles displayed differences in polymorphic behav-
18
ior depending on the type of triglyceride and

Transition enthalpy [J/g]

phospholipid employed for stabilization (Fig. 4).
The fraction of metastable a-modification was gener-
ally larger in dispersions of longer-chain triglycerides
as reflected in a strong melting endotherm in most
dispersions. In contrast, in trilaurin—and some of the
trimyristin—systems the presence of a-modification
could be deduced only from the occurrence of an
exothermic transition in the range of the a-transition.
Stabilization with natural soybean lecithin led if at all
to the formation of only very small transitions point-
ing to the presence of a-form. In contrast, DPPC and
hydrogenated soybean lecithin led to pronounced a-
transitions, in particular in tripalmitin and tristearin
dispersions. Stabilization with egg lecithin or DMPC
produced intermediate behavior. The progress of poly-
morphic transitions was monitored by DSC over one
week for cold stored trimyristin dispersions and for
tripalmitin and tristearin nanoparticles stored at room
temperature. During storage the thermal events due to
the a-modification became smaller or disappeared
depending on the composition of the sample and the
transition enthalpy of the stable h-polymorph in-
creased. The progress of polymorphic transitions
was very fast in trimyristin nanoparticles. Indepen-
dently of the stabilizer composition the enthalpy value
for the h-melting transition in these dispersions was
around 16 J/g already 1 h after controlled crystalliza-
tion and did not change notably during the monitored
period. In tripalmitin and tristearin dispersions the
amount of h-modification evolved more gradually in
spite of the higher storage temperature and there was
an obvious dependence on the type of phospholipid
(Fig. 5). The polymorphic transitions were slowest in
tristearin nanoparticles, in particular when these were
16
14
12
10
8
6
4
2
Tristearin (C18)
0
0 1 2 3 4 5 6 7
Days after preparation
Fig. 5. Evolution of the transition enthalpy for the h-polymorph
during storage of tripalmitin and tristearin dispersions at room
temperature. Selected tripalmitin samples (open symbols) were
also investigated after cold storage (cs). The first measurement
was performed after 1 h of storage. Dotted lines between data points
are drawn to guide the eye. The dashed line at the top of each
diagram indicates the transition enthalpy measured for the bulk
material in the h-form normalized to the same concentration as in
the dispersions. For clarity, the values for D116-S75/SGC, D116-
S75-3/SGC and D116-E100/SGC are not displayed. The data for
D116-DPPC/SGC after 7 days of storage could not be evaluated
quantitatively.
stabilized with the long-chain saturated phospholipids
DPPC and S100-3. Also E100 led to a comparatively
slow transformation of tristearin nanoparticles. In the
DPPC- and S100-3-containing tristearin dispersions
which displayed very strong a-transitions immediate-
ly after preparation, the a-modification was still pre-
dominant after about one year of cold storage (~ 65 or
70% of the triglyceride, respectively). After the same
period of storage at 23 8C, the fraction of residual a-
polymorph was lower, but still significant (~ 9 or
25%, respectively). Measurements on selected tripal-
mitin dispersions during the first week of storage also
revealed a slightly (S100) or distinctly (E100, S100-3)
 H. Bunjes, M.H.J. Koch / Journal of Controlled Release 107 (2005) 229–243
slower transition in cold stored samples reflected by a
lower melting enthalpy of the h-polymorph compared
to samples stored at room temperature (Fig. 5).
To compare the effect of the saturated long-chain
phospholipid DPPC with that of S100 during crystal-
lization, corresponding trimyristin samples were in-
vestigated in continuous, temperature dependent X-
ray diffraction measurements (Fig. 6). The dispersion
stabilized with the aid of S100 contained a large
fraction of h-modification already 20 min after the
onset of crystallization. In contrast, the sample con-
taining DPPC remained almost completely in the a-
modification during the course of the experiment. In
this sample, the first small angle reflection pointing to
the crystallization of triglyceride was detected at a
considerably higher temperature (~18 8C) than in the
sample stabilized with S100 (~11 8C) in agreement
with DSC results. Moreover, the diffraction pattern of
the dispersion with DPPC displayed a weak reflection
in the wide angle range already around 25–26 8C, i.e.,
well before the onset of triglyceride crystallization. In
contrast, the small and wide angle reflections
appeared virtually simultaneously in the S100-con-
taining dispersion. The position of the weak reflection
in the wide angle range of D114-DPPC/SGC corre-
sponded to that of the a-modification of the triglyc-
eride. In addition, a very broad, diffuse scattering peak
could be detected in the small angle scattering curves
of this dispersion soon after the start of the cooling
S100
β
°C
~ 2.5
~ 5.5
~ 8.5
α
~ 10
~ 13
1.5 2.0 2.5
s [1/nm]
Fig. 6. Wide angle X-ray diffraction patterns obtained during crystallization of trimyristin nanoparticles stabilized with the aid of S100 or DPPC,
respectively, from the melt. The time points refer to the first appearance of a small angle reflection which was considered to mark the onset of
crystallization.
237
program (already above 40 8C) which had no analogue
in the scattering curves of the D114-S100/SGC disper-
sion. Comparable wide angle reflections and some small
angle scattering effects were also observed in D112-
emulsions containing DPPC and S100-3 as well as in a
high-pressure homogenized dispersion of pure DPPC.
Corresponding emulsion systems stabilized with the aid
of S100 and E100 were comparable to D114-S100/SGC
prior to crystallization in X-ray scattering.
5. Discussion
Dispersion series prepared with the same triglycer-
ide and the different phospholipids gave similar par-
ticle sizes in PCS. In particular, there was no reduction
in dispersion quality when the samples contained
saturated or egg phospholipids. Negative effects of
the alternative phospholipids on colloidal stability
were also not detected by laser diffraction. The
cause for the increase in viscosity and gelation ten-
dency observed for the cold stored S100-3- and
DPPC-containing D116 dispersions is not yet clear,
but the instability does not seem to be inherent to the
stabilization regime as it was neither found for the
corresponding systems stored at room temperature nor
for dispersions of other triglycerides containing these
phospholipids. Saturated phospholipids also appear to
be less susceptible to the chemical reactions leading to
α
DPPC
min °C
+ 40 ~ 6
+ 30 ~ 9
+ 20 ~ 12
+ 10 ~ 15
+5 ~ 16.5
-5 ~ 19.5
3.0 1.5 2.0 2.5 3.0
s [1/nm]
238 H. Bunjes, M.H.J. Koch / Journal of Controlled Release 107 (2005) 229–243
discoloration observed in dispersions containing nat-
ural phospholipids, especially those with egg lecithin.
The different types of phospholipid employed in
the stabilizer mixture lead, however, to major differ-
ences in the T C and polymorphic transitions of the
triglyceride nanoparticles. All triglycerides reach their
lowest T C upon stabilization with the most unsaturat-
ed soybean lecithin (cf. Table 1 for phospholipid
composition). In previous investigations, the T C of
soybean lecithin/sodium glycocholate stabilized tri-
palmitin nanoparticles was shown to be at the lower
limit of T Cs for tripalmitin dispersions stabilized with
different types of surfactants [6]. Such behavior is
usually regarded as indicative of homogeneous nucle-
ation [16]. It seems thus justified to assume that there
is very little if any specific influence of this stabilizer
composition on the T C and that crystallization in
S100-containing dispersions is caused by homoge-
neous nucleation. Use of egg yolk lecithin, which
contains a much higher fraction of saturated fatty
acids already increases the T C for most triglycerides
except tristearin. As observed earlier, egg lecithin also
enhances crystallization of trimyristin in gel-forming
dispersions stabilized without additional cosurfactant
[17]. Fully saturated phospholipids, particularly those
with longer fatty acid chains, have an even larger
effect on the T C of most dispersed triglycerides. The
influence on the T C is specific for the different tri-
glyceride/phospholipid combinations: the T C of the
short-chain triglyceride trilaurin is increased com-
pared to the S100-containing dispersion to almost
the same value by all other phospholipids investigated
here. In contrast, triglycerides with longer chains than
trilaurin require fully saturated phospholipids with
long-chain fatty acid residues to obtain the highest
increase in T C. The longer the triglyceride chain, the
stronger the requirement for long, saturated phospho-
lipid chains to induce a large effect. For tripalmitin
and tristearin dispersions only the fully saturated,
long-chain soybean phospholipids, which contain
about 85% stearoyl chains, lead to the highest T C.
Phospholipid-stabilized emulsions do not seem to
have been studied in this context yet, but induction of
droplet crystallization at higher temperatures by some
emulsifiers has been observed previously in disper-
sions of lipidic material and was attributed to surface
heterogeneous nucleation [6,16,18]. Among the po-
tential causes considered for this phenomenon in
Tween stabilized hexadecane emulsions [16],
bcrystallizationQ of the emulsifier molecules in the
particle interface is supposed to be the predominant
mechanism in most dispersions with increased T C
studied here. The surfactant layer of the triglyceride
nanoparticles consists predominantly of phospholi-
pids, presumably with incorporated bile salt mole-
cules. The thermal precrystallization event which is
always observed upon stabilization with saturated
phospholipids is most likely caused by a phase tran-
sition involving rigidification of the phospholipid
chains which also occurs in the triglyceride-free dis-
persions. The occurrence of a single X-ray reflection
in the wide angle range in dispersions stabilized with
saturated long-chain phospholipids prior to crystalli-
zation of the triglyceride (which would be character-
ized by both, small and wide angle reflections)
confirms this assumption as it points to the formation
of short-range order. The solidified phospholipid
chains could act as a template for surface heteroge-
neous nucleation causing the increase in T C.
The temperature range of precrystallization in the
triglyceride dispersions is often comparable to that of
the thermal transition in the corresponding triglycer-
ide-free system but in other cases starts already quite
significantly above that of the pure phospholipid/bile
salt dispersion, e.g., in tripalmitin and tristearin dis-
persions containing DMPC and S100-3. A direct
comparison between the phospholipid transitions in
triglyceride-free and triglyceride-containing disper-
sions is, however, difficult. The phase transition tem-
perature of dispersed phospholipids depends on
different parameters, inter alia on the size of the
structures formed. Although the preparative condi-
tions used here for the triglyceride-free phospholipid
dispersions and the triglyceride dispersions were sim-
ilar the state of the phospholipids in the two types of
dispersions are likely to be different. In the triglycer-
ide dispersions the phospholipids are presumably par-
titioned between the stabilizer layer of the triglyceride
particles and vesicles, potentially also mixed micelles,
formed by excess phospholipid in the aqueous phase.
The triglycerides can affect the phase transition of the
phospholipid as suggested by the occasionally very
significant differences between the phospholipid
phase transition temperatures in triglyceride-free sys-
tems and the precrystallization event in the
corresponding triglyceride dispersions. Small amounts
 H. Bunjes, M.H.J. Koch / Journal of Controlled Release 107 (2005) 229–243
of triglycerides can be solubilized in phospholipid
bilayers [19] and are also supposed to penetrate into
the phospholipid monolayer stabilizing the surface of
triglyceride emulsion droplets to a certain extent [20].
The assumption of a specific interaction of triglycer-
ide and phospholipid leading to a modified crystalli-
zation behavior in some of the dispersions studied
here is supported by the fact that the transition en-
thalpy during precrystallization often appears to be
much larger than to be expected from the behavior of
the pure phospholipid/bile salt dispersions.
In the present case, it is assumed that the saturated
phospholipids at the surface of the emulsion droplets
and in vesicles solidify at some point during cooling
of the dispersion. During this phase transition, small
amounts of triglyceride incorporated in the phospho-
lipid bi- or surface monolayers may cosolidify as
shown for tripalmitin incorporated into DPPC bilayers
[19]. In some cases the interaction with the triglycer-
ide leads to a higher transition temperature for the
phospholipid. In the emulsion droplets, the solidified
stabilizer layer induces crystallization of the triglyc-
eride core by acting as a nucleation site.
This mechanism is, however, not applicable for
dispersions stabilized with egg lecithins. These phos-
pholipids enhance crystallization for most of the dis-
persed triglycerides but no precrystallization event is
observed. In this case, a btemplatingQ effect of liquid
surfactant tails may be invoked as a potential mech-
anism for the effect on the T C [16]. The preferential
orientation of the hydrophobic surfactant chains in the
particle interface might impose a certain degree of
order also on the neighboring triglyceride molecules.
As surfactant chains with a high molecular similarity
to the dispersed oil are considered most effective in
this mechanism [16], the differences between egg and
soybean lecithin would be attributed to a higher sim-
ilarity (more saturated chains in egg lecithin) between
the acyl chains in egg lecithin and in the dispersed
triglycerides. Alternatively, the effect could be
explained by a phenomenon similar to that proposed
for surfactant stabilized lipid emulsion droplets con-
taining hydrophobic additives such as sucrose oligoe-
sters, diglycerides or polyglycerol esters [21–24].
Addition of even small amounts of these hydrophobic
surface active agents with long, saturated acyl resi-
dues increases the T C of the oil phase compared to the
additive-free system. Small molecular aggregates of
239
these additives are assumed to crystallize within the
surfactant layer of the emulsion droplets and act as a
template for oil crystallization [25]. Formation of
inverse micelles of the hydrophobic additives in the
oil phase may have an additional contribution to
heterogeneous nucleation upon solidification of the
surfactant chains upon cooling. Transferred to the
triglyceride systems containing egg phospholipids,
the saturated acyl chains of the phospholipids may
form small clusters in the interfacial layer which could
solidify upon cooling and induce crystallization of the
triglyceride core. Whether inverse phospholipid
micelles dispersed in the triglyceride droplets might
contribute to the increase in T C remains unclear. Al-
though phospholipids are able to associate into inverse
micelles in liquid triglycerides and the presence of
inverse micelles in solidified lipid nanoparticle matri-
ces has been suggested [26] it is usually found that
excess phospholipid not required for interfacial stabi-
lization partitions into the water phase in aqueous
triglyceride–phospholipid dispersions [27, 28]. As
the phospholipid was processed from the aqueous
phase during preparation of the dispersions studied
here, partitioning of a relevant fraction into the tri-
glyceride phase is not expected.
From the practical point of view, the increase in T C
is particularly important for the trilaurin dispersions.
In technical processes it does not seem feasible to
crystallize trilaurin particles with low crystallization
tendency like those stabilized with S100 by simply
cooling the dispersions to subzero temperatures since
the risk of uncontrolled crystallization of the aqueous
phase increases with the sample volume. Hitherto,
flash freezing or freeze drying have been used for
solidification of triglyceride nanoparticles with poor
crystallization tendency [29,30]. This requires, how-
ever, time consuming and costly additional processing
steps. Moreover, the freezing step is regarded as a
major risk for the colloidal quality of solid lipid
nanoparticles [31] and should thus be avoided. With
the use of saturated phospholipids in the stabilizer
composition the crystallization problem of trilaurin
nanoparticles can be overcome without the need or
risk of freezing the aqueous phase.
In addition to their effect on the T C, the different
phospholipids also influence the polymorphic behav-
ior of the nanoparticles. In saturated monoacid trigly-
cerides, the metastable a-modification is usually
240 H. Bunjes, M.H.J. Koch / Journal of Controlled Release 107 (2005) 229–243
formed upon crystallization from the melt. Transfor-
mation into the stable h-form can either be induced by
heating the system to the melting point of the a-
modification or proceeds below the a-melting point
upon aging of the sample as a result of the monotropic
phase behavior. As established earlier for bulk trigly-
cerides [32] shorter-chain triglycerides undergo the
transition upon aging more easily than long-chain
ones because their activation energy for transforma-
tion is smaller. This explains why the a-polymorph is
barely detectable in trilaurin and trimyristin disper-
sions whereas it is more stable in dispersions of longer
chain triglycerides in good agreement with previous
investigations [2]. Moreover, the transition rate of a
given triglyceride increases with decreasing tempera-
ture difference between storage temperature and a-
melting point [32] as also observed here for some
tripalmitin and tristearin dispersions stored at different
temperatures.
Compared to natural soybean lecithin longer-chain,
saturated phopholipids and also egg lecithin exert a
retarding effect on the polymorphic transitions in spite
of the fact that the corresponding nanoparticles gen-
erally crystallize at higher temperature, i.e. closer to
the a-melting point. This retarding effect, which is
particularly pronounced for DPPC and fully hydroge-
nated soybean phospholipids, was clearly observed in
the DSC studies on freshly crystallized nanoparticles
as well as in the temperature-resolved X-ray diffrac-
tion study of trimyristin dispersions. The retarding
effects persist during storage of the tripalmitin and
tristearin dispersions. For the trimyristin dispersions,
differences during storage could hardly be detected
due to the very rapid transformation process in this
comparatively short-chain triglyceride already upon
cold storage. Handling at room temperature during
sample preparation for DSC measurements may
have led to an additional loss of residual a-form in
this dispersion.
The effect of surface active agents on the polymor-
phism of bulk triglycerides has already been studied in
some detail due to its impact on food systems. In
contrast, much less is known about the influence on
triglyceride polymorphism in dispersed systems, in
particular for surfactants having a low oil solubility
in the presence of water and with regard to effects on
storage. In saturated bulk triglycerides, particularly
certain hydrophobic, saturated long-chain surfactants
were observed to delay transformations into more
stable polymorphs [33]. The effect was attributed to
the incorporation of the surfactant molecules into the
triglyceride crystal lattice and specific interactions of
the triglyceride with the hydrophilic headgroup of the
surfactant. The changes in polymorphic behavior of
palm oil emulsions containing a hydrophobic food
emulsifier were also attributed to effects of the emul-
sifier within the droplet [34]. It cannot completely be
ruled out that trace amounts of phospholipid might
migrate into the bbulkQ of the triglyceride nanoparticles
and interact with the triglyceride molecules in a similar
way. It is, however, unlikely that such bbulk effectQ
would predominate in the nanoparticulate systems
investigated here since the phospholipids should be
preferentially localized at the interface and in the
aqueous phase as discussed above. The effect may
thus rather be caused by phospholipid molecules
trapped at the particle interface. Interfacial processes
have also been made responsible for the effects of very
hydrophilic, water-soluble surfactants and their mix-
tures with phospholipids on the polymorphism of tri-
glyceride nanoparticles [8]. Studies on emulsified
triglycerides or alkanes containing hydrophobic food
emulsifiers as additive also point to the role of the
interfacial layer for the influence of the additive on the
polymorphism of the dispersed phase. In most cases,
the effect could simply be attributed to an increase of
the T C above the melting point of the metastable a-
form due to surface heterogenous nucleation on inter-
facial templates leading to the formation of the hT-form
instead [22, 23]. Recently, however, the formation of
unusual polymorphic forms was reported for disper-
sions of even numbered alkanes as the result of addi-
tion of hydrophobic food emulsifiers. The formation of
these polymorphs which had previously only been
known for uneven numbered alkanes was attributed
to specific interaction of the alkane molecules with
additive templates formed in the droplet interface [35].
For the saturated long-chain phospholipids in the pres-
ent study the formation of a bshellQ of solidified phos-
pholipid chains, presumably with some incorporated
triglyceride, around the nanoparticles already prior to
crystallization can be deduced from the DSC and X-
ray data. After crystallization of the triglyceride in the
a-form this solidified shell might suppress the poly-
morphic transformation as it may not easily provide
the bworking spaceQ and molecular mobility required
 H. Bunjes, M.H.J. Koch / Journal of Controlled Release 107 (2005) 229–243
for the rearrangements occurring during transforma-
tion. In contrast, a more bfluid-likeQ state is expected
for the surface of nanoparticles stabilized with highly
unsaturated phospholipids and may also occur with
saturated phospholipids above their chain-melting
temperature. Such a fluid-like state of the surfactant
layer is expected to enhance the transition as it would
provide mobility not only for the fluid chains of the
phospholipid but probably also for some of the tri-
glyceride chains. Accelerating effects observed for
liquid oils and surfactants on bulk triglycerides and
liquefied drug on triglyceride nanoparticles have been
explained in a similar way [33,36–38]. The assump-
tion of a fluidized interface can explain the much
more rapid transformation for the soybean-lecithin
stabilized dispersion in comparison to the DPPC
and S100-3 stabilized systems, which both contain
solidified chains at the temperature of storage. The
situation is more complex for the DMPC and, in
particular, egg lecithin stabilized dispersions which
display intermediate behavior. In the DMPC-contain-
ing dispersions, the interfacial layer may at least be
partially fluidized at a storage temperature (23 8C)
around the chain-melting temperature of the pure
phospholipid. In dispersions containing egg lecithin,
the phospholipid layer would be expected to be com-
pletely fluidized as the phase transition temperature of
these phospholipids is much below room temperature.
The polymorphic transitions observed with egg phos-
pholipids are, however, rather slow. Tentatively, very
long saturated chains of these phospholipids (e.g. of
arachidic acid) might interact with the triglyceride
molecules and restrict their mobility but the effect
remains largely unexplained at present. In the discus-
sion of the effects of phospholipids on the poly-
morphic transformation it should be kept in mind
that they will probably be modulated by the presence
of glycocholate molecules in the interfacial layer. This
surfactant is known to induce a very slow transfor-
mation in triglyceride nanoparticles when used as the
only stabilizer [8,39].
From the pharmaceutical point of view, there are
two main aspects of the pronounced effect of the
phospholipid type on the polymorphism of the matrix
triglyceride: particles in the a-form can undergo
polymorphic transformation into the thermodynami-
cally stable h-modification upon storage. This will
lead to changes in important properties such as par-
241
ticle shape, density and, most probably, also drug
incorporation capacity and/or intraparticular distribu-
tion. Gradual changes in related important pharma-
ceutical properties of the dispersion like viscosity,
drug solubilization and/or release behavior during
shelf life would be unacceptable for a pharmaceutical
product. Consequently, these products would have to
be designed in such a way that either exclusively the
thermodynamically stable form is present after pro-
duction or that the stability of the metastable parti-
cles is so high that polymorphic transitions cannot
take place within shelf life. The formation of only
the thermodynamically stable form can be achieved
by thermal treatment during or after preparation of
the nanoparticles. Aging the particles at a tempera-
ture close to the melting point of the respective a-
modification, for example, will lead to rapid trans-
formation into the more stable form. Additives, such
as liquid oils or other liquid-like lipidic substances
which are known to accelerate polymorphic transi-
tions may also be used but at the cost of an increased
complexity of the system. Reliable stabilization of
the a-form over a pharmaceutically relevant period
(e.g., at least six months) under realistic storage
conditions would be a very interesting option due
to the expected modified properties of a-form parti-
cles, e.g., an increased drug loading capacity due to a
lower packing density of this modification. This task
remains, however, a challenge and none of the sys-
tems under investigation here would meet the
requirements. Even the comparatively stable S100-3
and DPPC containing tristearin dispersions form a
considerable fraction of h-form particles already
upon cold storage. The combination of saturated
long-chain phospholipid/bile salt as stabilizer with
even longer-chain triglycerides and/or the inclusion
of mixed long-chain triglycerides might be promising
approaches to further improve the a-form stability.
6. Conclusion
Stabilization of triglyceride nanoparticles with the
aid of phospholipids containing long, saturated fatty
acid chains lowers the supercooling tendency of the
dispersed triglyceride and increases its T C by several
degrees compared to stabilization with soybean leci-
thin. Saturated phospholipids for stabilization produce
242 H. Bunjes, M.H.J. Koch / Journal of Controlled Release 107 (2005) 229–243
a thermal event prior to triglyceride crystallization
upon cooling, which is attributed to the solidification
of the phospholipid chains in the emulsifier layer,
probably with some contribution of triglyceride. For
trilaurin which often poses recrystallization problems
after preparation by melt-homogenization shorter-
chain saturated phospholipids like DMPC or partially
unsaturated egg lecithin are already very effective in
increasing the T C. The increase in T C of trilaurin
nanoparticles stabilized with the aid of adequate phos-
pholipids allows one to completely crystallize these
particles at 0 8C without freezing the aqueous phase.
The colloidal quality of the dispersions is not com-
promised by the use of saturated or egg phospholi-
pids. Phospholipids with saturated acyl chains appear
to be advantageous with regard to the chemical sta-
bility of the stabilizer system.
The polymorphic transitions of all triglycerides are
retarded in the presence of saturated and egg lecithin
compared to soybean lecithin. Tentatively, this may be
due to spatial restrictions and loss of fluidity at the
particle surface and/or a templating effect of the sur-
factant layer. In tristearin dispersions containing hy-
drogenated soybean lecithin or DPPC the a-
modification is very stable upon cold storage. For
future investigations, the possibility to prepare parti-
cles with low transformation rate opens interesting
possibilities to study the influence of polymorphic
form also on other important nanoparticle properties
such as colloidal stability or drug incorporation ca-
pacity, localization within the particles and release.
The information concerning parameters causing re-
tarded polymorphic transitions may lead to new for-
mulations with enhanced a-form stability under
pharmaceutically relevant conditions in the future.
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